Rational design and synthesis of a novel anti-leukemic agent targeting Bruton's tyrosine kinase (BTK), LFM-A13 [alpha-cyano-beta-hydroxy-beta-methyl-N-(2,5-dibromophenyl)propenamide]

In a systematic effort to design potent inhibitors of the anti-apoptotic ty rosine kinase BTK (Bruton's tyrosine kinase) as anti-leukemic agents with a poptosis-promoting and chemosensitizing properties, we have constructed a t hree-dimensional homology model of the BTK kinase domain. Our modeling stud ies revealed a distinct rectangular binding pocket near the hinge region of the BTK kinase domain with Leu(460), Tyr(476) Arg(525), and Asp(539) resid ues occupying the corners of the rectangle. The dimensions of this rectangl e are approximately 18 x 8 x 9 x 17 Angstrom, and the thickness of the pock et is approximately 7 Angstrom. Advanced docking procedures were employed f or the rational design of leflunomide metabolite (LFM) analogs with a high likelihood to bind favorably to the catalytic site within the kinase domain of BTK. The lead compound LFM-A13, for which we calculated a K-i value of 1.4 mu M, inhibited human BTK in vitro with an IC50 value of 17.2 +/- 0.8 m u M. Similarly, LFRIA13 inhibited recombinant BTK expressed in a baculoviru s expression vector system with an IC50 value of 2.5 mu M, The energeticall y favorable position of LFM-A13 in the binding pocket is such that its arom atic ring is close to Tyr(476), and its substituent group is sandwiched bet ween residues Arg(525) and Asp(539). In addition, LFM-A13 is capable of fav orable hydrogen bonding interactions with BTK via Asp(539) and Arg(525) res idues. Besides its remarkable potency in BTK kinase assays, LFM-A13 was als o discovered to be a highly specific inhibitor of BTK, Even at concentratio ns as high as 100 mu g/ml (similar to 278 mu M), this novel inhibitor did n ot affect the enzymatic activity of other protein tyrosine kinases, includi ng JAK1, JAK3, HCK, epidermal growth factor receptor kinase, and insulin re ceptor kinase, In accordance with the anti-apoptotic function of BTK, treat ment of BTK+ B-lineage leukemic cells with LFM-A13 enhanced their sensitivi ty to ceramide- or vincristine-induced apoptosis, To our knowledge, LFM-A13 is the first BTK-specific tyrosine kinase inhibitor and the first anti-leu kemic agent targeting BTK.